Autism spectrum disorder (ASD) is a highly heritable, but genetically complex, neurodevelopmental disorder. Increasing evidence points to an interaction between genetic vulnerability and environmental risk factors in the generation of ASD. Human genetics and animal models suggest that alterations in synapse formation and maintenance may be fundamental to the etiology of ASD, and recent human exome-profiling studies suggest transcription and chromatin remodeling functions may be affected. Epigenetic regulation of gene transcription, including through developmentally dynamic and cell type-specific patterns, is a plausible mechanism mediating long-term environmental contributions to ASD. Environmental impacts on the overall configuration of DNA methylation (methylome) may lead to aberrant silencing or activation of genes involved brain circuitry maturation, with subsequent functional and behavioral consequences. Recently, the first analysis of whole- genome single-base resolution methylome maps of developing frontal cortex in mice and humans revealed extensive methylome reconfiguration during development from fetal to young adult. Importantly, a specific form of cytosine methylation, in non-CG sites, accumulated preferentially in neurons, coinciding with the period of synaptogenesis in both species. These results point to a potential role of the neuronal methylome in healthy development of neural circuits that could be particularly vulnerable to pathological disruption. Leveraging on these data, as well as on preliminary data showing dynamic changes in CG methylation patters during the transition between embryonic and early life in mouse brain, this proposal will test the hypothesis that alteration of specific forms of DNA methylation are involved in the origins of ASD. To test this hypothesis, Aim 1 will produce single-base resolution methylome maps in the two major neuronal populations in frontal cortex i.e. excitatory and inhibitory neurons, in a well-established model of ASD, the maternal immune activation (MIA) model. Additionally, to test for additive effects of environmental exposures in a compromised gestation, Aim 2 will expose the MIA animals to the environmental toxin PBDE. Finally, Aim 3 will analyze the methylome changes produced by MIA in animals that are deficient for the autism risk gene Shank3 to test for gene x environment interactions. The transcriptional consequences of methylome changes will be assessed by RNA- Seq for each neuronal population at each time-point during development, and long term behavioral consequences will be assessed through a battery of behavioral tests relevant to ASD. New, sophisticated computational analysis procedures will be used to integrate diverse and large-scale empirical data sets to provide a powerful and stringent test of our hypotheses.